Structures and biological evaluation of phenylpropanoid derivatives from Murraya koenigii

Article abstract of DOI:10.1016/j.bioorg.2019.01.038

Four new phenylpropanoid derivatives (1–4), together with eleven known analogues (5–15) were isolated and identified by comparison with their references and extensive spectroscopic methods from Murraya koenigii for the first time. Compounds (1–15) were assayed for their inhibitory activities by measuring IL-6-induced STAT3 promoter activities in HepG2 cells, and found compounds 1, 2, 6, and 15 showed inhibitory effects with IC₅₀ values of 11.5, 18.7, 8.9, and 22.7 μM, respectively. The inhibitory activities of compounds (1–15) were screened against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, and found compounds 3, 4, 9, 11, and 14 exhibited inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC₅₀ values of 32.7, 7.9, 42.1, 58.9, and 62.4 μM, respectively.

Full text of DOI:10.1016/j.bioorg.2019.01.038

BioorganicChemistry86(2019)159–165
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structures and biological evaluation of phenylpropanoid derivatives from
Murraya koenigii
⁎
⁎
⁎
Qinge Maa,1, , Rongrui Weia,1, , Ming Yanga, , Xiaoying Huanga, Guoyue Zhonga, Zhipei Sangc,
Jianghong Dongd, Jicheng Shua, Jianqun Liua, Rui Zhanga, Jianbo Yangb, Aiguo Wangb,
⁎
Tengfei Jib, Yalun Sub,
a State Key Laboratory of Innovative Drugs and High Efficiency Energy Saving and Consumption Reduction Pharmaceutical Equipment, Key Laboratory of Modern
Preparation of TCM of Ministry of Education, Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional
Chinese Medicine, Nanchang 330004, China
b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
c College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China
d College of Chemistry and Pharmaceutical Engineering, Huanghuai University, Zhumadian 463000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Four new phenylpropanoid derivatives (1–4), together with eleven known analogues (5–15) were isolated and
identified by comparison with their references and extensive spectroscopic methods from Murraya koenigii for
the first time. Compounds (1–15) were assayed for their inhibitory activities by measuring IL-6-induced STAT3
promoter activities in HepG2 cells, and found compounds 1, 2, 6, and 15 showed inhibitory effects with IC50
values of 11.5, 18.7, 8.9, and 22.7 μM, respectively. The inhibitory activities of compounds (1–15) were screened
against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, and found compounds
3, 4, 9, 11, and 14 exhibited inhibitions against LPS-induced NO production in RAW264.7 macrophages, with
IC50 values of 32.7, 7.9, 42.1, 58.9, and 62.4 μM, respectively.
Murraya koenigii
Rutaceae
Phenylpropanoid derivatives
Biological evaluation
1. Introduction
a result, four new phenylpropanoid derivatives (1–4), and eleven
known analogues (5–15) were isolated and identified from the active
Murraya koenigii is a favorite condiment from the Rutaceae family. It
is mainly distributed in the tropical and subtropical regions [1]. The
leaves, stems, and roots of this plant have been served as folk medicine
for treating various diseases [2]. Phytochemical investigations of M.
koenigii have revealed a wide array of natural products including al-
kaloids [3], sesquiterpenes [4], volatile oils [5], and alkenes [6]. Dif-
ferent parts of M. koenigii have varied biological activities such as anti-
inflammatory [7], anti-oxidative [6], nephroprotective [8], hepato-
protective [4], anti-listerial [9], and PTP1B inhibitory [3] activities.
After consulting a large of references, there were no reports on in-
hibitory activities by measuring IL-6-induced STAT3 promoter activities
in HepG2 cells and inhibition against lipopolysaccharide (LPS)-induced
NO production in RAW264.7 macrophages of M. koenigii. With the aim
of identifying new bioactive compounds from M. koenigii, we analyzed
the different extraction sites by carrying out a bioassay-guided in-
vestigation of M. koenigii in order to evaluate its further bioactivities. As
fraction (EtOAc soluble fraction) of M. koenigii for the first time.
Described herein are the isolation, structural elucidation, and bioac-
tivity evaluation of compounds (1–15) from M. koenigii in this work.
2. Materials and methods
2.1. General experimental procedures
The optical rotations were recorded on a Perkin-Elmer 241 digital
polarimeter at 20 ℃. The melting points were measured by a XT5B
microscopic melting point apparatus which was uncorrected. The CD
spectra were recorded on a JASCO J-815CD spectrometer. The UV and
IR spectra were measured by a Hitachi UV-240 spectrophotometer and
a Nicolet 5700 FTIR microscope spectrometer with KBr pellets, re-
spectively [4]. The 1D and 2D NMR measurements were performed on
Varian Mercury-300, Inova-501, DD2-500, Bruker Avance III HD-600
⁎ Corresponding authors.
E-mail addresses: maqinge2006@163.com (Q. Ma), weirongrui2011@163.com (R. Wei), yangming16@126.com (M. Yang), suyalun@imm.ac.cn (Y. Su).
1 Co-first author.
https://doi.org/10.1016/j.bioorg.2019.01.038
Received 5 October 2018; Received in revised form 8 January 2019; Accepted 21 January 2019
Availableonline24January2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

Q. Ma et al.
BioorganicChemistry86(2019)159–165
spectrometers with tetramethylsilane as an internal standard. The ESI-
MS and HR-ESI-MS data were measured by a Q-Trap LC/MS/MS spec-
trometer and an Agilent 1100 series LC/MSD Trap SL mass spectro-
meter. The HPLC separation was performed on an Agilent 1200 series
with a DIKMA (4.6 × 250 mm) analytical column packed with C18
(5 μm) and the preparative HPLC was conducted by a Shimadzu LC-6AD
instrument with a SPD-20A detector and an YMC-Pack ODS-A column
(250 × 20 mm, 5 μm) [6]. The column chromatography was performed
with Sephadex LH-20, Toyopearl HW-40, and silica gel (160–200 mesh,
200–300 mesh). The TLC was carried out on precoated silica gel GF254
plates and the spots were visualized under UV light (254 or 365 nm) or
by spraying with 10% sulfuric acid in EtOH followed by heating [10].
2.4. IL-6-induced STAT3 luciferase assay
Interleukin-6 (IL-6) is involved in a broad spectrum of inflammatory
diseases, it has been postulated to be an effective therapy in the pa-
thogenesis of several inflammatory diseases [12]. These diseases are
commonly characterized by excessive IL-6 levels that lead to the in-
duction of IL-6 signaling cascades, resulting in activation of the Janus
kinase (Jak)/signal transducer and activator of transcription (STAT)
pathways in the intracellular environment [13]. STAT3, one of the
STAT family proteins, functions as a signal transducer in the cytoplasm
and as a transcription factor in the nucleus [14]. STAT3-regulated genes
are closely related to the production of pro-inflammatory cytokines or
enzymes that cause the above-mentioned inflammatory diseases [15].
The HepG2 cells were put in Dulbecco's modified Eagle's medium
(10% fetal bovine serum) and were cultured in the 96-well culture plate
(5 × 104 cells/mL) with 5% CO2 at 37 °C for 24 h. The HepG2 cells were
exposed to a variety of concentrations of compounds (1–15), and these
cells were incubated for an hour with IL-6 (10 ng/mL). After 6 h, the
HepG2 cells were washed with PBS and treated with a lysis buffer
(50 μL) for 15 min, followed by intermittent shaking at room tem-
perature. The cell lysate (25 μL) was transferred to a white microtiter
plate, and 50 μL of a luciferase assay reagent (Promega, Madison, WI)
was mixed into each well. The luciferase activity was measured by a
luminometer. The HepG2 cells were measured using a MTT assay which
incubated in a 96-well plate for 24 h and treated with compounds
(1–15) at the indicated concentrations for 48 h [16].
2.2. Plant material
Murraya koenigii was harvested from Xi Shuang Ban Na Tropical
Botanical Garden, the Chinese Academy of Sciences, Kunming, China in
August 2010. This plant was identified by Prof. Lin Ma (Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College). A voucher specimen (No. ID-S-2436) was de-
posited at the Herbarium of Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking Union Medical College, Beijing
100050, China [4].
2.3. Extraction and isolation
The air-dried Murraya koenigii (22.50 kg) was extracted three times
with 95% EtOH (60 L) of heating under reflux for 3 h each time. The
solvent was concentrated to afford 2.10 kg of dark crude extract, which
was successively partitioned with CHCl3 (3 × 6000 mL), EtOAc
(3 × 6000 mL), and n-butanol (3 × 6000 mL) to yield three fractions:
CHCl3 soluble fraction (33.50 g), EtOAc soluble fraction (24.90 g), and
n-butanol soluble fraction (221.20 g), respectively [11]. According to
the screening results of bioactivity-guided investigation, the EtOAc
soluble fraction exhibited potential inhibitory activities by measuring
IL-6-induced STAT3 promoter activities in HepG2 cells and inhibition
against lipopolysaccharide (LPS)-induced NO production in RAW264.7
macrophages.
2.5. Inhibitory assay of NO production
The RAW264.7 macrophages were cultured in 24-well plates (105
cells/well), and they were maintained in a water-saturated atmosphere
of 5% CO2 at 37 °C. These cells were co-incubated with drugs and LPS
(1 μg/mL) for 24 h. The amount of NO was assessed by determined the
nitrite concentration in the cultured RAW264.7 macrophage super-
natants with Griess reagent. Aliqueots of supernatants (100 μL) were
incubated with 1% sulphanilamide (50 μL), 1% naphthylethylenedia-
mine (50 μL) in 2.5% phosphoric acid solution, sequencely. The ab-
sorbances of compounds (1–15) were read using a microtiter plate
reader at 570 nm [17].
The EtOAc soluble fraction was chromatographed over silica gel
(200–300 mesh, 100 g, 3.0 × 60 cm) eluting with a gradient elution
(petroleum ether/acetone = 10:1 → 1:1 → 1:5, v/v) to yield three
fractions: SMJY-B (4.20 g), XMJY-Y (10.50 g), and SMJY-Y (8.80 g). The
SMJY-Y fraction was chromatographed over Sephadex LH-20 eluting
with 90% MeOH to give two sub-fractions: SMJY-Y-a (2.35 g) and
SMJY-Y-b (3.10 g). The separation of SMJY-Y-a was separated by MPLC
(25–40% MeOH) and preparative HPLC (detection at 203 nm, 6 mL/
min), successively, yielded 1 (7.34 mg, purity > 96% by HPLC), 2
(8.14 mg, purity > 97% by HPLC), and 5 (5.08 mg, purity > 96% by
HPLC). The separation of SMJY-Y-b was separated by MPLC (20–40%
MeOH) and preparative HPLC (detection at 205 nm, 6 mL/min), suc-
3. Results and discussion
3.1. Spectroscopic data
(7′E,8S)-9′-hydroxy-7′-propen-3′,5′-dimethoxyphenyl-3-methox-
yphenyl-7,9-propanediol-4-O-β-D-glu-copyranoside
(1):
colourless
powder; [α]2D0 −11.20 (c 0.30, MeOH); mp 214.3–215.5 °C; UV
(MeOH) λmax: 203 and 274 nm; IRνmax 3372.3, 1660.9, 1598.9, and
1501.5 cm−1
;
1H and 13C NMR spectroscopic data see Table 1; HR-ESI-
MS: m/z 575.2112 [M+Na]+ (calcd. for C27H36NaO12, 575.2099).
(7R)-2,6-dimethoxyphenyl-7,9-propanediol-1-O-β-D-glucopyranoside
(2): colourless powder; [α]2D0 –6.10 (c 0.12, MeOH); mp 184.2–185.6 °C;
cessively, yielded
6 (9.22 mg, purity > 95% by HPLC) and 14
(51.46 mg, purity > 96% by HPLC). In a similar way, the XMJY-Y
fraction was also chromatographed over Sephadex LH-20 eluting with
95% MeOH to give two sub-fractions: XMJY-Y-a (3.70 g) and XMJY-Y-b
(4.05 g). The separation of XMJY-Y-a was separated by MPLC (30–40%
MeOH) and preparative HPLC (detection at 204 nm, 6 mL/min), suc-
cessively, yielded 3 (6.22 mg, purity > 96% by HPLC), 7 (4.37 mg,
purity > 97% by HPLC), 8 (6.21 mg, purity > 95% by HPLC), 9
(4.24 mg, purity > 97% by HPLC), 10 (4.25 mg, purity > 95% by
HPLC), 11 (5.21 mg, purity > 96% by HPLC), and 15 (14.30 mg,
purity > 96% by HPLC). The separation of XMJY-Y-b was separated by
MPLC (35–45% MeOH) and preparative HPLC (detection at 204 nm,
6 mL/min), successively, yielded 4 (6.26 mg, purity > 96% by HPLC),
12 (5.33 mg, purity > 97% by HPLC), and 13 (2.47 mg, purity > 96%
by HPLC). The structures of compounds (1–15) were shown in Fig. 1.
UV (MeOH) λmax: 203 nm; IRνmax 3369.0, 1642.3, 1597.8, and
1509.3 cm−1
;
1H and 13C NMR spectroscopic data see Table 1; HR-ESI-
MS: m/z 413.1425 [M+Na]+ (calcd. for C17H26NaO10, 413.1418).
(2′R,4′R,7S)-2′,4-dihydroxy-3-methoxyphenyl-4′-hydroxymethyl-
tetrahydro-1H-pyran-1-one (3): colourless crystal; [α]2D0 +5.50 (c 0.02,
MeOH); mp 128.4–129.3 °C; UV (MeOH) λmax: 204, 230, and 281 nm;
IRνmax 3392.8, 1763.1, 1603.5, and 1518.3 cm−1
;
1H and 13C NMR
spectroscopic data see Table 2; HR-ESI-MS: m/z 291.0854 [M+Na]+
(calcd. for C13H16NaO6, 291.0839).
(1R,10S)-1-hydroxy-7-(10-hydroxybutyl)-2,3-dihydrobenzofuran-
8(6H)-one (4): yellowish oil; [α]2D0 +4.65 (c 0.15, MeOH); UV (MeOH)
λ
max: 202 and 274 nm; IRνmax 3365.1 and 1737.8 cm−1
;
1H and 13C
NMR spectroscopic data see Table 2; HR-ESI-MS: m/z 247.0946 [M
+Na]+ (calcd. for C12H16NaO4, 247.0941).
160

Q. Ma et al.
BioorganicChemistry86(2019)159–165
Fig. 1. Structure of compounds 1–15.
3.2. Structure determination
single peak at δH 6.75 (2H, s, H-2′/6′) in the aromatic field (Table 1).
Moreover, a set of typical signals at δH 6.58 (1H, d, J = 15.9 Hz, H-7′),
6.27 (1H, dt, J = 15.9, 6.0 Hz, H-8′), and 4.22 (2H, d, J = 76.0 Hz, H-
9′), which revealed a trans-propenol group in compound 1, it was
connected to aromatic ring indicated the phenylpropanoid skeleton
[18] was in compound 1. A characteristic doublet at δH 4.86 (1H, d,
J = 7.5 Hz, H-1″) was ascribed to the anomeric proton of glucosyl unit,
corresponding to δC 105.3 (C-1″) of the 13C NMR and HSQC spectra
(Table 1), which indicated the presence of the glucose was in compound
1. The fragments of compound 1 was determined by the HMBC corre-
lations of H-2/C-4, C-7; H-5/C-1; H-6/C-7; H-7/C-9; H-8/C-4′; H-7′/C-
Compound 1 was isolated as a colorless powder. Its HR-ESI-MS data
showed a sodium adduct ion at m/z 575.2112 [M+Na]+, establishing
the molecular formula as C27H36O12, indicating 10 degrees of un-
saturation. The IR spectrum showed absorption bands for a hydroxyl
group (3372.3 cm−1) and an aromatic ring group (1660.9, 1598.9,
1501.5 cm−1) and its UV spectrum exhibited at λmax: 203 and 274 nm.
The 1H NMR data of compound 1 indicated the presence of the
1,3,4-trisubstituted benzene system at δH 6.72 (1H, d, J = 1.5 Hz, H-2),
6.97 (1H, d, J = 7.5 Hz, H-5), 6.97 (1H, dd, J = 7.5, 1.5 Hz, H-6) and a
161

Q. Ma et al.
BioorganicChemistry86(2019)159–165
Table 1
aglycone 1a [19]. The negative cotton effect at 274 nm was observed in
the CD spectrum of compound 1a, which was induced by reagent of
Rh2(OCOCF3)4. According to the spiral rule and literature [20], the
absolute configuration of C-8 was determined as 8S. Therefore, com-
1H NMR (300 MHz, CD3OD), 13C NMR(125 MHz, CD3OD), and key HMBC
correlations of compounds 1 and 2.
No.
1
2
pound
1 was identified as (7′E,8S)-9′-hydroxy-7′-propen-3′,5′-di-
δH
δC
HMBC
–
δH
δC
HMBC
methoxyphenyl-3-methoxyphenyl-7,9-propanediol-4-O-β-D-glu-copy-
ranoside.
1
2
3
4
5
6
7
–
129.9
–
135.2
154.2
–
–
Compound 2 was acquired as a colorless powder with a rotation of
[α]2D0 −6.10 (c 0.12, MeOH). Its molecular formula was determined to
be C17H26O10 on the basis of the HR-ESI-MS: m/z 413.1425 [M+Na]+
(calcd. for C17H26NaO10 413.1418) and NMR spectroscopic data
6.72(d,1.5)
114.2 C-4,C-7
–
–
145.6
135.7
–
–
6.73(s)
104.7 C-1
–
–
143.5
–
6.97(d,7.5)
6.97(dd,7.5,1.5)
3.42(m)
116.5 C-1
112.2 C-7
6.73(s)
–
104.7 C-1
154.2
–
(Table 1). According to the spectroscopic data of UV (MeOH) λmax
:
55.8
C-9
4.78(dd,8.1,3.0) 72.3
C-3,C-
203 nm and IRνmax 3369.0, 1642.3, 1597.8, 1509.3 cm−1, it was con-
cluded that compound 2 was a derivative of phenylpropanoid [18].
The 1H NMR spectrum of compound 2 showed a single peak at δH
6.73 (2H, s, H-3/5) in the aromatic field. A characteristic glucosyl unit
was found at δH 4.84 (1H, d, J = 7.5 Hz, H-1′) and δC 105.5 (C-1″) in
the 1H NMR and 13C NMR spectra (Table 1), which indicated the pre-
sence of the glucose was in compound 2. In the middle of 1H NMR
spectrum, a 1,3-propanediol unit was found at δH 4.78 (1H, dd, J = 8.1,
3.0 Hz, H-7), 1.92 (2H, m, H-8), 3.72 (1H, m, H-9a), 3.63 (1H, m, H-9b),
corresponding to δC 72.3 (C-7), 42.9 (C-8), 60.0 (C-9) of the 13C NMR
and HSQC spectra (Table 1). The fragments of compound 2 were con-
nected by some key correlations of H-3/C-1; H-5/C-1; H-7/C-3, C-5, C-
9; H-1′/C-1, C-3′; H-3′/C-5′; H-4′/C-6′ in the HMBC spectrum (Fig. 2)
and some key correlations of H-7/H-8; H-8/H-9 in the 1H-1H COSY
spectrum (Fig. 2). Compound 2 was hydrolyzed to afford the aglycone
2a [19]. The positive cotton effect at 273 nm and the negative cotton
effect at 327 nm were observed in the CD spectrum of compound 2a.
The absolute configuration of C-7 in compound 2 was determined as 7R
according to the spiral rule and literature [20]. Consequently, com-
pound 2 was identified as (7R)-2,6-dimethoxyphenyl-7,9-propanediol-
1-O-β-D-glucopyranoside.
5,C-9
8
5.60(dd,9.6,5.7)
3.86(m)
3.79(m)
–
88.8
64.9
64.9
132.8
C-4′
1.92(m)
3.72(m)
3.63(m)
4.84(d,7.5)
42.9
60.0
60.0
–
–
–
9a
9b
1′
–
–
–
105.5 C-1,C-
3′
2′
6.75(s)
104.4
154.5
140.0
154.5
104.4
104.4
–
–
–
–
–
–
3.44(m)
3.21(m)
3.43(m)
3.42(m)
3.81(m)
3.68(m)
–
75.7
78.3
71.3
77.8
62.6
62.6
–
–
3′
–
C-5′
C-6′
–
4′
–
5′
–
6′a
6′b
7′
6.75(s)
6.75(s)
6.58(d,15.9)
–
–
131.9 C-2′, C-
6′, C-9′
–
8′
9′
1″
6.27(dt,15.9,6.0) 127.7
–
–
–
–
–
–
–
–
–
–
–
4.22(d,6.0)
4.86(d,7.5)
63.8
105.3 C-4,C-
3″
2″
3.49(m)
2.27(m)
3.43(m)
3.47(m)
3.83(m)
3.68(m)
–
75.7
78.3
71.3
77.8
62.6
62.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3″
C-5″
–
–
4″
C-6″
–
–
5″
–
–
–
–
–
–
–
–
–
6″a
–
–
6″b
–
–
2-OCH3
6-OCH3
3.87(s)
3.87(s)
–
56.9
56.9
–
–
–
Compound 3 was obtained as a colorless crystal with a molecular
formula of C13H16O6, which was determined based on HR-ESI-MS at m/
z 291.0854 (calcd for C13H16O6Na, 291.0839), indicating 6 degrees of
unsaturation. Its UV spectrum showed absorptions at λmax: 204, 230,
281 nm and IR absorptions indicated the existence of hydroxyl
(3392.8 cm−1), carbonyl (1763.1 cm−1), aromatic ring (1603.5,
1518.3 cm−1) functional groups.
3′-OCH3 3.83(s)
5′-OCH3 3.91(s)
57.0
56.8
–
Table 2
1H NMR (300 MHz, CD3COCD3), 13C NMR(125 MHz, CD3COCD3), and key
HMBC correlations of compounds 3 and 4.
The 1H NMR spectrum of compound 3 showed an ABX system at δH
7.04 (1H, d, J = 1.8 Hz, H-2), 6.82 (1H, d, J = 8.1 Hz, H-5), 6.88 (1H,
dd, J = 8.1, 1.8 Hz, H-6). Moreover, a methoxyl signal at δH 3.85 (3H,
s) and a double peak at δH 4.68 (1H, d, J = 6.3 Hz, H-7) were observed
in the middle of 1H NMR spectrum of compound 3 (Table 2). Ad-
ditionally, a saturated cyclohexanone [δC 179.1 (C-1′), 70.6 (C-2′), 46.9
(C-3′), 49.1 (C-4′), 70.8 (C-5′)] was determined by its molecular for-
mula, degrees of unsaturation, and NMR spectrum of compound 3 [21].
The relative structure of compound 3 was determined by the HMBC
correlations of H-2/C-4; H-5/C-1; H-7/C-2, C-3′; H-2′/C-4′; H-5′/C-1′
(Fig. 2) and the 1H-1H COSY correlations of H-5/H-6; H-2′/H-3′; H-3′/
H-4′; H-4′/H-7 (Fig. 2). The absolute configurations of C-2′, C-4′ in
compound 3 were determined by the CD method. The 7-OH of com-
pound 3 was protected by tert-butyldimethylsilyl chloride (TBSCl) in
order to eliminate the interference of measuring its CD spectrum, and
afforded the derivative 3a. The negative cotton effect at 256 nm was
observed in the CD spectrum of compound 3a, which was induced by
reagent of Rh2(OCOCF3)4. The absolute configurations of C-2′, C-4′ in
compound 3 were confirmed as 2′R, 4′R according to the octant rule of
lactone and reference [22]. Meanwhile, the absolute configuration of C-
7 in compound 3 was determined as 7S by the correlation between 7-
OH/2′-OH and 7-OH/H-4′ in the 2D-NOESY spectrum and the Cahn-
Ingold-Prelog order rule [23]. Hence, compound 3 was identified as
(2′R,4′R,7S)-2′,4-dihydroxy-3-methoxyphenyl-4′-hydroxymethyl-tetra-
hydro-1H-pyran-1-one.
No.
3
4
δH
δC
HMBC
–
δH
δC
HMBC
1
–
132.5
3.99(m)
2.05(m)
2.55(m)
5.39(t,15.6,7.8)
2.22(d,6.0)
–
70.4
C-7
C-6
C-5
–
2
7.04(d,1.8)
110.6 C-4
24.7
3
–
148.4
147.3
–
–
17.7
4
–
112.3
153.9
149.6
126.9
169.2
28.6
5
6.82(d,8.1)
115.6 C-1
–
6
6.88(dd,8.1,1.8) 119.8
–
–
7
4.68(d,6.3)
87.0
C-2,C-3′
–
–
8
–
–
–
–
–
9
–
–
–
2.33(m)
4.28(m)
C-8
–
10
11
12
1′
2′
3′a
3′b
4′
5′a
5′b
–
–
–
65.0
–
–
–
1.49(dd,14.7,7.2) 22.9
0.95(dd,14.7,7.2) 14.0
C-12
C-9
–
–
–
–
–
179.1
70.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.22(m)
C-4
–
–
4.36(dd,9.6,2.1) 46.9
4.03(dd,9.0,3.3) 46.9
3.54(dd,9.0,3.6) 49.1
4.54(dd,9.6,6.9) 70.8
–
–
–
–
–
C-1′
C-1′
–
–
4.28(m)
70.8
56.2
–
3-OCH3 3.85(s)
–
2′, C-6′, C-9′; H-1″/C-4, C-3″; H-3″/C-5″; H-4″/C-6″ (Fig. 2) and the
1H-1H COSY correlations of H-5/H-6; H-7/H-8; H-7′/H-8′; H-8′/H-9′
(Fig. 2). The absolute configuration of C-8 in compound 1 was de-
termined by the CD method. Compound 1 was hydrolyzed to afford the
Compound 4 was obtained as a yellowish oil, the molecular formula
162

Q. Ma et al.
BioorganicChemistry86(2019)159–165
Fig. 2. Key HMBC and 1H-1H COSY correlations of compounds 1–4.
of compound 4 was determined as C12H16O4 by HR-ESI-MS showed [M
+Na]+ at m/z 247.0946 [M+Na]+ (calcd. for C12H16NaO4,
247.0941), indicating 5 degrees of unsaturation. Its UV spectrum dis-
played absorbance at λmax: 202 and 274 nm, and its IR spectrum
showed the absorption bands of hydroxyl (3365.1 cm−1) and carbonyl
(1737.8 cm−1) functionalities.
(Fig. 2) and some key correlations of H-1/H-2; H-2/H-3; H-3/H-4; H-9/
H-10; H-10/H-11 in the 1H-1H COSY spectrum (Fig. 2). The absolute
configurations of C-1 in compound 4 was determined as 1R on the basis
of biosynthetic pathway and reference [25]. In the same way, the 1-OH
of compound
4 was protected by tert-butyldimethylsilyl chloride
(TBSCl) in order to eliminate the interference of measuring CD spec-
trum of C-10, and afforded the derivative 4a. The positive cotton effect
at 232 nm and the negative Cotton effect at 277 nm were observed in
the CD spectrum of compound 4a, which were induced by reagent of
Rh2(OCOCF3)4. The absolute configurations of C-10 in compound 4
were confirmed as 10S according to the octant rule and reference [25].
So, compound 4 was determined as (1R,10S)-1-hydroxy-7-(10-hydro-
xybutyl)-2,3-dihydrobenzofuran-8(6H)-one.
A typical triplet at δH 5.39 (1H, t, J = 15.6, 7.8 Hz, H-4) and three
saturated carbon signals at δC 70.4 (C-1), 24.7 (C-2), 17.7 (C-3) were
found in the NMR spectra of compound 4, which revealed the typical
characteristic of the cyclohexene skeleton [24]. In the middle of 1H
NMR spectrum, two typical multiple peaks at δH 3.99 (1H, m, H-1), 4.28
(1H, m, H-10), corresponding to δC 70.4 (C-1), 65.0 (C-10) of the 13C
NMR and HSQC spectra (Table 2), which indicated two oxygenate
carbons were in compound 4. The fragment of –CH2CH3 at δH 1.49 (2H,
dd, J = 14.7, 7.2 Hz), 0.95 (3H, t, J = 14.7, 7.2 Hz) were observed in
the high of 1H NMR spectrum, corresponding to δC 22.9 (C-11), 14.0 (C-
12) of the 13C NMR and HSQC spectra (Table 2). The fragments of
compound 4 were connected by some key correlations of H-1/C-7; H-2/
C-6; H-3/C-5; H-9/C-8; H-10/C-12; H-11/C-9 in the HMBC spectrum
Additionally, eleven known compounds (5–15) belonging to phe-
nylpropanoid derivatives, which were isolated and identified as (1′R)-4-
O-β-D-glucopyranoside-3,5-dimethoxyphenyl-1′-propanol (5) [26], ci-
trusin B (6) [27], (7′R,8′R,8E)-4′,7′-dihydroxy-3′-methoxyphenyl-8′-
hydroxymethyl-ethoxy-3,5-dimethoxyphenyl-8-propenoic acid methyl
ester (7) [28], (7S,8S)-1′-hydroxy-3′,5′-dimethoxyphenoxy-4-hydroxy-
163

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BioorganicChemistry86(2019)159–165
Table 3
Inhibitory effects of compounds (1–15) on IL-6-induced STAT3 activation.a
Compound
IC50 (μM)
Compound
IC50 (μM)
1
2
3
4
5
6
7
8
11.5
1.1
3.8
9
> 100
> 100
> 100
> 100
> 100
> 100
18.7
10
> 100
> 100
> 100
11
12
13
8.9
6.8
14
> 100
> 100
15
22.7
24.5
5.4
1.9
Genisteinb
a
Values are mean
SD of three experiments, with each data point done in
triplicate.
b
Genistein was used as the positive control.
3-methoxyphenyl-7,9-propanediol (8) [29], (7′E,7S,8S)-9′-hydroxy-7′-
propen-3′-methoxyphenyl-4-hydroxy-3-methoxyphenyl-7,9-propane-
diol (9) [30], (1S,2R)-4,4′-hydroxy-3,3′-methoxyphenyl-1,3-propane-
diol (10) [31], (7S,8R)-4,4′-dihydroxy-3,3′-dimethoxyphenyl-7-ethoxy-
9-propanol (11) [32], (7S,8R)-4-hydroxy-3-methoxyphenyl-7,8,9-pro-
panetriol (12) [33], (7S,8R)-4-hydroxy-3,5-dimethoxyphenyl-7,8,9-
propanetriol (13) [34], lariciresinol-4-O-β-D-glucopyranoside (14) [35],
and (7S,7′S,8S,8′R)-4,4″,7′,9′-Tetrahydroxy-3,3′,3″-trimethoxyphenyl-
7,9-propanediol (15) [36] by comparison of their physical and spec-
troscopic data with those reported in the references. To the best of our
knowledge, the known compounds (5–15) were isolated from this plant
for the first time.
Fig. 3. Inhibitory activities of selected compounds against LPS-induced NO
production.
activation of IL-6-induced STAT3 in HepG2 cells with genistein as the
positive control. The HepG2 cells stably expressing pSTAT3-luciferase
were excited with IL-6 (10 ng/mL) for 6 h in the presence of the test
compounds (1–15), and pSTAT3-inducible luciferase activity was then
measured [16]. Compounds 1, 2, 6, and 15 displayed important in-
hibitory effects, with IC50 values of 11.5, 18.7, 8.9, and 22.7 μM, re-
spectively, and genistein was used as the positive control
(IC50 = 24.5 μM). The other compounds exhibited weak or no in-
hibitory effects (IC50 > 100 μM) (Table 3).
The inhibitory activities of compounds (1–15) were screened
against NO production in lipopolysaccharide (LPS)-induced RAW264.7
macrophage cells [38]. As shown in Table 4, compounds 3, 4, 9, 11,
and 14 showed certain inhibitions against LPS-induced NO production
in RAW264.7 macrophages, with IC50 values of 32.7, 7.9, 42.1, 58.9,
and 62.4 μM, respectively. Dexameth was used as positive control with
an IC50 value of 8.5 μM. Among them, compound 4 showed obvious
inhibitions against LPS-induced NO production in RAW264.7 macro-
phages, and compound 3, 9, 11, and 14 showed moderate inhibitions
against LPS-induced NO production in RAW264.7 macrophages. How-
ever, the other compounds exhibited no inhibitions against LPS-induced
NO production in RAW264.7 macrophages with IC50 > 100 μM
(Fig. 3).
3.3. Acid hydrolysis of compounds 1, 2, 5, 6, and 14
Compounds 1, 2, 5, 6, and 14 (5 mg each) were treated in 5% HCl
(0.5 mL) and heated at 90 °C for 2 h, respectively [37]. After cooling,
each reaction mixture was extracted with EtOAc, and the aqueous layer
was neutralised with 0.1 M NaOH. As a result, the glucoses were ob-
tained from compounds 1, 2, 5, 6, and 14, which were detected by thin
layer chromatography (TLC) with authentic sugars. The type of glucose
was identified by TLC method with authentic sugar [4].
3.4. Statistical analysis of biological activities
Compounds (1–15) were evaluated for their ability to inhibit the
Table 4
Inhibitory activities of (1–15) against LPS-induced NO production in RAW264.7 macrophages.a
Compound
Inhibition (%)
IC50(μM)
80 μM
–
60 μM
40 μM
20 μM
10 μM
dexamethasoneb
–
–
–
–
8.5
1
41.37
38.59
79.47
82.33
36.76
46.25
50.15
39.08
67.24
41.19
60.74
37.09
42.22
56.44
42.16
0.38
0.24
0.12
0.36
0.15
0.82
0.63
0.22
0.28
0.15
0.24
0.33
0.10
0.48
0.52
30.12
28.10
71.08
76.39
33.28
36.73
34.22
31.21
59.08
34.10
53.71
33.23
39.48
46.13
37.13
0.17
0.19
0.30
0.24
0.38
0.50
0.58
0.13
0.16
0.17
0.84
0.17
0.13
0.77
0.44
26.24
21.77
64.11
70.26
27.55
31.64
30.88
28.42
48.50
29.44
42.56
26.44
28.80
37.35
33.27
0.73
0.13
0.65
0.79
0.84
0.34
0.60
0.54
0.13
0.18
0.71
0.52
0.36
0.64
0.47
23.36
19.29
38.28
61.85
20.23
24.65
26.35
20.25
35.07
22.25
23.54
21.30
24.62
30.54
28.42
0.35
0.32
0.33
0.29
0.33
0.31
0.16
0.33
0.23
0.55
0.14
0.45
0.17
0.57
0.35
17.02
12.38
21.05
53.76
19.22
22.60
24.19
16.62
26.21
20.77
22.59
18.09
19.44
23.18
20.41
0.52
0.49
0.54
0.29
0.66
0.14
0.25
0.11
0.53
0.58
0.16
0.53
0.33
0.42
0.27
> 100
> 100
32.7
2
3
4
7.9
5
> 100
> 100
> 100
> 100
42.1
6
7
8
9
10
11
12
13
14
15
> 100
58.9
> 100
> 100
62.4
> 100
a
b
NO concentration of control group: 2.12
Positive control.
0.11 μM, NO concentration of LPS-treated group: 42.30
0.22 μM.
164

Q. Ma et al.
BioorganicChemistry86(2019)159–165
4. Conclusion
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Standard Revision Research Project of National Pharmacopoeia
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